Our experiments have revealed the existence of a hitherto unknown, trans-acting, macromolecular regulatory system controlling the location and relative activity of mammalian recombination hotspots by activating, suppressing and modulating the activity of specific hotspots. Genetic variation of this regulatory system enabled its discovery and now provides a means of identifying its components and their interactions, an essential first step in understanding its relevance to issues of human genetics as well as questions of population biology and evolution. The locations of hotspots and their relative activity in humans and mice (our principal mammalian research model organism) determine patterns of linkage disequilibrium and the possibilities for closely linked genes to be coinherited, important issues in efforts to identify genes important in human health and disease. The first regulatory gene we identified, Prdm9, determines hotspot locations by activating recombination there; it has no quantitative modifiers, and its role is independent of gene dosage. Here we will investigate the complementary regulatory system controlling quantitative levels of hotspot activity, a matter of equal concern in elucidating the structure of this novel regulatory system and its ability to influence inheritance patterns. We will study the quantitative regulation of three hotspots whose activities are regulated by genes that differ between the mouse strains C57BL/6J and CAST/EiJ; two have their activity reduced by the presence of CAST alleles, and one has its activity enhanced. We will do so by applying a new, considerably improved, quantitative assay system for hotspot activities in sperm samples that relies on high throughput DNA sequencing; mapping and cloning the regulatory genes involved; testing their interactions with each other; determining whether they act in a dose dependent manner, indicating whether they act catalytically or stoichimetrically, and finally, testing whether they control the rate of initiation of recombination or the decision between the alternative recombination pathways leading to crossing over or the formation of non-crossover gene conversions. We expect to learn whether each hotspot has its own unique regulatory system or there are shared regulatory elements, whether up and down regulation are effected by the same genes, whether controls are exerted on the initiation of recombination or the choice between alternate pathways of recombination, and the manner in which any of these genes interact with each other. These data together with the molecular identity of these genes will likely provide important clues as to their mechanism of action. The answers to these questions will considerably enhance our understanding of one of the most basic biological processes, genetic recombination. PUBLIC HEALTH RELEVANCE: Genetic recombination is the biological process that enables us to screen human populations for genes determining human health and disease. The proposed experiments will enhance our ability to carry out such studies by increasing our understanding of how recombination is regulated. The findings are applicable to every human condition that is genetically influenced, including all of our major diseases.